Assay for detecting and quantifying hiv-1

ABSTRACT

Method of detecting HIV-1 nucleic acids using nucleic acid amplification and a molecular torch hybridization probe. The invented method is characterized by high levels of precision in the quantitation of HIV-1 targets at low copy numbers, and by accurate detection of different HIV-1 subtypes, including M group and O group variants.

RELATED APPLICATIONS

This application in a continuation of U.S. application Ser. No.11/240,046, filed Sep. 30, 2005, now pending, which claims the benefitof U.S. Provisional Application No. 60/615,533, filed Sep. 30, 2004. Theentire disclosures of these prior applications are hereby incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology. Morespecifically, the invention relates to diagnostic assays for detectingand quantifying the nucleic acids of HIV-1.

BACKGROUND OF THE INVENTION

Advances in the clinical management of individuals infected with thehuman immunodeficiency virus type 1 (HIV-1) have been able to reduceviral titers below the detection limits of some early-generation HIV-1assays. More specifically, highly active anti-retroviral drug therapy(HAART) can reduce the viral load down to a level approaching 50 HIV-1RNA copies/ml, a level substantially below the 400-500 copies/mlthreshold of some previous detection assays. This fact, together with adesire to monitor and maintain low viral titers, necessitated thedevelopment of improved quantitative assays for measuring HIV-1 RNA.(Elbeik et al., J. Clin. Micro. 38:1113-1120 (2000)) Complicatingmatters, however, is the fact that useful quantitative assays must becapable of accurately measuring a range of genetically diverse HIV-1variants.

Three classes of HIV-1 have developed across the globe: M (major), O(outlying) and N (new). Among the M group, which accounts for greaterthan 90% of reported HIV/AIDS cases, viral envelopes have diversified sogreatly that this group has been subclassified into nine major cladesincluding A-D, F-H, J and K, as well as several circulating recombinantforms. Subtypes within the HIV-1 O group are not clearly defined, andthe diversity of sequences within the O group is nearly as great as thediversity of sequences in the HIV-1 M group. Phylogenetic analyses ofthe gag and env genes have failed to reveal clades of O group viruses asclearly as the clades detected in the M group. Subtypes and sub-subtypesof the HIV-1 M group are thought to have diverged in humans following asingle chimpanzee-to-human transmission event. In contrast, the HIV-1 Oand N groups are each thought to have resulted from separatechimpanzee-to-human transmission events. Of the completely sequencedHIV-1 genomes, nearly 20% have a mosaic structure consisting of at leasttwo subtypes, yet another potential complication for ultrasensitiveHIV-1 detection assays. (Spira et al., J. Antimicrobial Chemotherapy51:229 (2003).)

Most viral load monitoring is currently performed in the developedWestern World where the Glade B (i.e., “subtype B” hereafter), whichrepresents only about 3% of HIV infections worldwide, predominates.Importantly, the HIV-1 viral subtypes are expanding in differentgeographical regions, thereby imposing an additional requirement forbroad detection capacity on detection and viral load monitoring assays.Accordingly, there is a need for ultrasensitive HIV-1 detection assayswhich are capable of accurately measuring the full range of HIV-1subtypes. The present invention addresses this need.

An example quantitative HIV-1 assay, performed using real-timemonitoring of a nucleic acid amplification reaction, has been describedin published International Patent Application WO 2003106714.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to a reaction mixture useful foramplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleicacids. The invented reaction mixture ordinarily includes first andsecond amplification primers. The first amplification primer includes afirst primer target-hybridizing sequence that can independentlyhybridize to a first strand of HIV-1 M group nucleic acids, and to afirst strand of HIV-1 O group nucleic acids. The second amplificationprimer includes a second primer target-hybridizing sequence thathybridizes to an enzymatic extension product of the first amplificationprimer using as a template either the first strand of HIV-1 M groupnucleic acids or the first strand of HIV-1 O group nucleic acids. Thesecond primer target-hybridizing sequence consists essentially of SEQ IDNO:33. In a preferred embodiment, the second primer target-hybridizingsequence consists essentially of SEQ ID NO:2. When this is the case, thefirst primer target-hybridizing sequence may consist essentially of SEQID NO:13. Alternatively, the first primer target-hybridizing sequencemay consist essentially of SEQ ID NO:15. In a different preferredembodiment, the second primer target-hybridizing sequence consistsessentially of SEQ ID NO:5. When this is the case, the first primertarget-hybridizing sequence may consist essentially of SEQ ID NO:15. Inyet another preferred embodiment, the reaction mixture further includesa hybridization probe. In some instances, the hybridization probe is amolecular beacon hybridization probe or a molecular torch hybridizationprobe. Regardless of whether the hybridization probe is a molecularbeacon or a molecular torch, it is preferred in certain embodiments thatno more than two primers and a single probe are used for amplifying anddetecting the HIV-1 M group nucleic acids or the HIV-1 O group nucleicacids.

A second aspect of the invention relates to a method of quantifying thecombined amount of an HIV-1 M group nucleic acid and an HIV-1 O groupnucleic acid that may be present in a biological sample. The inventedmethod involves steps for: (a) combining in a single reaction vessel thebiological sample, a first amplification primer, a second amplificationprimer, and a hybridization probe; (b) amplifying, with substantiallyequal efficiency, any of the HIV-1 M group nucleic acid and the HIV-1 Ogroup nucleic acid present in the biological sample using an in vitroamplification reaction that relies on enzymatic extension of the firstamplification primer using a first strand of the HIV-1 M group nucleicacid or the HIV-1 O group nucleic acid as a first template to create afirst primer extension product, and enzymatic extension of the secondamplification primer using the first primer extension product as asecond template, whereby there are produced HIV-1 M group amplicons ifthe biological sample contained HIV-1 M group nucleic acids, and HIV-1 Ogroup amplicons if the biological sample contained HIV-1 O group nucleicacids; (c) monitoring amplicon production in the in vitro amplificationreaction as a function of time by a process that includes detection of asignal from the hybridization probe, whereby time-dependent quantitativedata is obtained; and (d) quantifying the combined amount of the HIV-1 Mgroup nucleic acid and the HIV-1 O group nucleic acid present in thebiological sample using the time-dependent quantitative data obtained inthe monitoring step. In accordance with this aspect of the invention,neither the first amplification primer nor the second amplificationprimer is fully complementary to the HIV-1 M group nucleic acid or thecomplement thereof, or to the HIV-1 O group nucleic acid or thecomplement thereof. Further in accordance with this aspect of theinvention, the hybridization probe hybridizes to both HIV-1 M groupamplicons and HIV-1 O group amplicons. Notably, the invented method alsois contemplated for use in detecting and quantifying HIV-1 N groupnucleic acids. In a preferred embodiment, the in vitro amplificationreaction is an isothermal in vitro amplification reaction that does notrequire temperature cycling to achieve some degree of exponentialamplification. More preferably, the isothermal in vitro amplificationreaction is a transcription associated amplification reaction that iseither a TMA reaction or a NASBA reaction. In an alternative preferredembodiment, the signal detected in the monitoring step is a fluorescentsignal, such as a fluorescent signal produced by a molecular torchhybridization probe. In a highly preferred embodiment, the firstamplification primer includes a first primer target-hybridizing sequencethat consists essentially of SEQ ID NO:15. More preferably, the secondamplification primer includes a second primer target-hybridizingsequence that consists essentially of SEQ ID NO:5. In accordance withanother preferred embodiment, no more than two primers and a singleprobe are used for amplifying and detecting both the HIV-1 M groupnucleic acid and the HIV-1 O group nucleic acid. In a highly preferredembodiment, the in vitro amplification reaction is an isothermal invitro amplification reaction. In an alternative highly preferredembodiment, the quantifying step involves comparing a quantitativeresult with no more than a single standard curve.

A third aspect of the invention relates to a method of establishing apoint on a standard curve that can be used for quantifying HIV-1 M groupnucleic acids and HIV-1 O group nucleic acids in a single reaction. Theinvented method involves steps for: (a) providing a known amount of anHIV-1 standard; (b) amplifying in an in vitro amplification reaction theHIV-1 standard using a first primer and a second primer in the presenceof a hybridization probe to produce HIV-1 standard amplicons, whereinthe amplification reaction amplifies HIV-1 M group nucleic acids andHIV-1 O group nucleic acids with substantially equal efficiency; (c)monitoring production of HIV-1 standard amplicons synthesized in the invitro amplification reaction as a function of time by a process thatinvolves detection of a signal from the hybridization probe, wherebyquantitative data is obtained; and (d) establishing from thequantitative data a point on the standard curve. In a preferredembodiment, the first amplification primer includes a first primertarget-hybridizing sequence that independently hybridizes to a firststrand of HIV-1 M group nucleic acids and to a first strand of HIV-1 Ogroup nucleic acids, wherein the second amplification primer includes asecond primer target-hybridizing sequence that hybridizes to anenzymatic extension product of the first amplification primer using as atemplate either the first strand of HIV-1 M group nucleic acids or thefirst strand of HIV-1 O group nucleic acids. In accordance with thisembodiment, (a) neither the first primer target-hybridizing sequence northe second primer target-hybridizing sequence is fully complementary toHIV-1 M group or HIV-1 O group nucleic acids or the complements thereof,and (b) the hybridization probe is able to hybridize either to HIV-1 Mgroup nucleic acids and HIV-1 O group nucleic acids, or to theircomplements. In one preferred embodiment, the hybridization probe is amolecular torch. More preferably, when the hybridization probe is amolecular torch, the HIV-1 standard is an HIV-1 M group nucleic acidstandard. Still more preferably, when the hybridization probe is amolecular torch, and when the HIV-1 standard is an HIV-1 M group nucleicacid standard, there can be an additional step for using the standardcurve to quantify an HIV-1 O group nucleic acid contained in abiological sample. In a different preferred embodiment, the HIV-1standard is an HIV-1 O group nucleic acid standard. When this is thecase, there can be a further step for using the standard curve toquantify an HIV-1 M group nucleic acid contained in a biological sample.In still another different embodiment, the in vitro amplificationreaction in the amplifying step can be an isothermal in vitroamplification reaction. When this is the case, the isothermal in vitroamplification reaction can be a transcription associated amplificationreaction, such as a TMA reaction or a NASBA reaction. In such aninstance, the step for monitoring can involve measuring a fluorescentsignal.

A fourth aspect of the invention relates to a method of preparing areaction mixture for amplifying either or both of HIV-1 M group nucleicacids and HIV-1 O group nucleic acids. The invented method includessteps for: (a) selecting a first amplification primer that includes asequence that independently hybridizes to a first strand of either HIV-1M group target nucleic acids or HIV-1 O group target nucleic acids; (b)selecting a second amplification primer that includes a sequence thathybridizes to enzymatic extension products of the first amplificationprimer using the first strand of either HIV-1 M group target nucleicacids or HIV-1 O group target nucleic acids as a template; (c) selectinga hybridization probe that hybridizes to amplicons synthesized by theuse of the first and the second amplification primers, wherein neitherthe first primer target-hybridizing sequence nor the second primertarget-hybridizing sequence is fully complementary to the HIV-1 M groupor HIV-1 O group nucleic acids or the complements thereof, and whereinthe first amplification primer, the second amplification primer, and thehybridization probe are further selected to amplify in an in vitroamplification reaction HIV-1 M group nucleic acids and HIV-1 O groupnucleic acids with substantially equal efficiencies; and (d) combiningin a single reaction vessel the first amplification primer, the secondamplification primer, and the hybridization probe. In a preferredembodiment, the reaction mixture includes no more than two primers and asingle hybridization probe for amplifying and detecting the HIV-1 Mgroup nucleic acids and HIV-1 O group nucleic acids. More preferably,the in vitro amplification reaction is an isothermal in vitroamplification reaction. Still more preferably, the isothermal in vitroamplification reaction is a transcription associated amplificationreaction that is either a TMA reaction or a NASBA reaction.

A fifth aspect of the invention relates to a composition for amplifyingHIV-1 M group target nucleic acids and HIV-1 O group target nucleicacids. The invented composition includes: (a) a first amplificationprimer that includes a first primer target-hybridizing sequence thatindependently hybridizes to a first strand of HIV-1 M group targetnucleic acids and to a first strand of HIV-1 O group target nucleicacids; and (b) a second amplification primer that includes a secondprimer target-hybridizing sequence that hybridizes to enzymaticextension products of the first amplification primer using the firststrand of either HIV-1 M group target nucleic acids or HIV-1 O grouptarget nucleic acids as a template. In accordance with this aspect ofthe invention, neither the first primer target-hybridizing sequence northe second primer target-hybridizing sequence is fully complementary tothe HIV-1 M group or HIV-1 O group target nucleic acids or thecomplements thereof. In a preferred embodiment, the composition alsoincludes a hybridization probe that hybridizes to an amplificationproduct produced in an in vitro amplification reaction by the combinedactivity of the first and second amplification primers using as atemplate either HIV-1 M group target nucleic acids or HIV-1 O grouptarget nucleic acids. More preferably, the composition amplifies HIV-1 Mgroup target nucleic acids and HIV-1 O group target nucleic acids in thein vitro nucleic acid amplification reaction with substantially equalefficiency. Still more preferably, the first primer target-hybridizingsequence consists essentially of SEQ ID NO:15, and the second primertarget-hybridizing sequence consists essentially of SEQ ID NO:5. In analternative preferred embodiment, the hybridization probe is a moleculartorch or a molecular beacon. In certain instances, it is preferred forthe hybridization probe to be a molecular torch. In other preferredembodiments, the second primer target-hybridizing sequence consistsessentially of SEQ ID NO:5. When this is the case, the first primertarget-hybridizing sequence may consist essentially of SEQ ID NO:15. Instill other preferred embodiments, when the composition includes theabove-mentioned hybridization probe, the first primer target-hybridizingsequence may consist essentially of SEQ ID NO:15, and the second primertarget-hybridizing sequence may consist essentially of SEQ ID NO:5. Morepreferably, the hybridization probe is a molecular torch.

A sixth aspect of the invention relates to a reaction mixture foramplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleicacids. The invented reaction mixture includes: (a) a first amplificationprimer that includes a first primer target-hybridizing sequence thatindependently hybridizes to a first strand of HIV-1 M group nucleicacids and a first strand of HIV-1 O group nucleic acids; (b) a secondamplification primer that includes a second primer target-hybridizingsequence that hybridizes to an enzymatic extension product of the firstamplification primer, using as a template either the first strand ofHIV-1 M group nucleic acids or the first strand of HIV-1 O group nucleicacids; and (c) a molecular torch hybridization probe that hybridizes toan amplicon synthesized by the combined activity of the firstamplification primer and the second amplification primer. In accordancewith this aspect of the invention, neither the first primertarget-hybridizing sequence nor the second primer target-hybridizingsequence is fully complementary to the HIV-1 M group or HIV-1 O groupnucleic acids or the complements thereof. Significantly, the HIV-1 Mgroup nucleic acids and HIV-1 O group nucleic acids amplify in thereaction mixture with substantially equal efficiency.

DEFINITIONS

The following terms have the following meanings for the purpose of thisdisclosure, unless expressly stated to the contrary herein.

As used herein, a “biological sample” is any tissue orpolynucleotide-containing material obtained from a human, animal orenvironmental sample. Biological samples in accordance with theinvention include peripheral blood, plasma, serum or other body fluid,bone marrow or other organ, biopsy tissues or other materials ofbiological origin. A biological sample may be treated to disrupt tissueor cell structure, thereby releasing intracellular components into asolution which may contain enzymes, buffers, salts, detergents and thelike.

As used herein, “polynucleotide” means either RNA or DNA, along with anysynthetic nucleotide analogs or other molecules that may be present inthe sequence and that do not prevent hybridization of the polynucleotidewith a second molecule having a complementary sequence.

As used herein, a “detectable label” is a chemical species that can bedetected or can lead to a detectable response. Detectable labels inaccordance with the invention can be linked to polynucleotide probeseither directly or indirectly, and include radioisotopes, enzymes,haptens, chromophores such as dyes or particles that impart a detectablecolor (e.g., latex beads or metal particles), luminescent compounds(e.g., bioluminescent, phosphorescent or chemiluminescent moieties) andfluorescent compounds.

A “homogeneous detectable label” refers to a label that can be detectedin a homogeneous fashion by determining whether the label is on a probehybridized to a target sequence. That is, homogeneous detectable labelscan be detected without physically removing hybridized from unhybridizedforms of the label or labeled probe. Homogeneous detectable labels arepreferred when using labeled probes for detecting HIV-1 nucleic acids.Examples of homogeneous labels have been described in detail by Arnoldet al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No.5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737. Preferred labelsfor use in homogenous assays include chemiluminescent compounds (e.g.,see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson et al., U.S. Pat.No. 5,658,737; and Arnold, Jr., et al., U.S. Pat. No. 5,639,604).Preferred chemiluminescent labels are acridinium ester (“AE”) compounds,such as standard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE,1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE,ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE).

A “homogeneous assay” refers to a detection procedure that does notrequire physical separation of hybridized probe from non-hybridizedprobe prior to determining the extent of specific probe hybridization.Exemplary homogeneous assays, such as those described herein, can employmolecular beacons or other self-reporting probes which emit fluorescentsignals when hybridized to an appropriate target, chemiluminescentacridinium ester labels which can be selectively destroyed by chemicalmeans unless present in a hybrid duplex, and other homogeneouslydetectable labels that will be familiar to those having an ordinarylevel of skill in the art.

As used herein, “amplification” or “amplifying” refers to an in vitroprocedure for obtaining multiple copies of a target nucleic acidsequence, its complement or fragments thereof.

By “target nucleic acid” or “target” is meant a nucleic acid moleculecontaining a target nucleic acid sequence.

By “target nucleic acid sequence” or “target sequence” or “targetregion” is meant a specific deoxyribonucleotide or ribonucleotidesequence comprising all or part of the nucleotide sequence of asingle-stranded nucleic acid molecule, and possibly comprising (whenspecified) the deoxyribonucleotide or ribonucleotide sequencecomplementary thereto. In general, a target nucleic acid sequence thatis to be amplified will be positioned between two oppositely disposedprimers, and will include the portion of the target nucleic acidmolecule that is partially or fully complementary to each of theprimers. In the context of the invention, a target nucleic acid moleculemay be, for example, an HIV-1 nucleic acid molecule. The portion of thistarget nucleic acid molecule to be amplified in an in vitro nucleic acidamplification reaction would be referred to as the “target nucleic acidsequence” to be amplified.

By “transcription associated amplification” is meant any type of nucleicacid amplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. One example of a transcriptionassociated amplification method, called “Transcription MediatedAmplification” (TMA), generally employs an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-template complementary oligonucleotide,and optionally may include one or more analogous oligonucleotides.Variations of TMA are well known in the art as disclosed in detail inBurg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos.5,399,491 and 5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras etal., PCT No. WO 88/01302; Gingeras et al., PCT No. WO 88/10315; Malek etal., U.S. Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCTNo. WO 95/03430. The methods of Kacian et al. are preferred forconducting nucleic acid amplification procedures of the type disclosedherein. Another example of a transcription associated amplificationmethod is the Nucleic Acid Sequence-Based Amplification (NASBA) methoddisclosed in U.S. Pat. No. 5,554,517.

As used herein, an “oligonucleotide” or “oligomer” is a polymeric chainof at least two, generally between about five and about 100, chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety that joins the subunits. Common nucleotidebase moieties are guanine (G), adenine (A), cytosine (C), thymine (T)and uracil (U), although other rare or modified nucleotide bases,including nucleotide analogs, able to hydrogen bond are well known tothose skilled in the art. Oligonucleotides may optionally includeanalogs of any of the sugar moieties, the base moieties, and thebackbone constituents. Preferred oligonucleotides of the presentinvention fall in a size range of about 10 to about 100 residues.Oligonucleotides may be purified from naturally occurring sources, butpreferably are synthesized using any of a variety of well knownenzymatic or chemical methods.

As used herein, a “hybridization probe” is an oligonucleotide thathybridizes specifically to a target sequence in a nucleic acid,preferably in an amplified nucleic acid, under conditions that promotehybridization, to form a detectable hybrid. A probe optionally maycontain a detectable moiety which either may be attached to the end(s)of the probe or may be internal. The nucleotides of the probe whichcombine with the target polynucleotide need not be strictly contiguous,as may be the case with a detectable moiety internal to the sequence ofthe probe. Detection may either be direct (i.e., resulting from a probehybridizing directly to the target sequence or amplified nucleic acid)or indirect (i.e., resulting from a probe hybridizing to an intermediatemolecular structure that links the probe to the target sequence oramplified nucleic acid). The “target” of a probe generally refers to asequence contained within an amplified nucleic acid sequence whichhybridizes specifically to at least a portion of a probe oligonucleotideusing standard hydrogen bonding (i.e., base pairing). A probe maycomprise target-specific sequences and optionally other sequences thatare non-complementary to the target sequence that is to be detected.These non-complementary sequences may comprise a promoter sequence, arestriction endonuclease recognition site, or sequences that contributeto three-dimensional conformation of the probe (e.g., as described inLizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). Sequences thatare “sufficiently complementary” allow stable hybridization of a probeoligonucleotide to a target sequence that is not completelycomplementary to the probe's target-specific sequence.

As used herein, an “amplification primer” is an oligonucleotide thathybridizes to a target nucleic acid, or its complement, and participatesin a nucleic acid amplification reaction. For example, amplificationprimers, or more simply “primers,” may be optionally modifiedoligonucleotides which are capable of hybridizing to a template nucleicacid and which have a 3′ end that can be extended by a DNA polymeraseactivity. In general, a primer will have a downstream HIV-1complementary sequence, and optionally an upstream sequence that is notcomplementary to HIV-1 nucleic acids. The optional upstream sequencemay, for example, serve as an RNA polymerase promoter or containrestriction endonuclease cleavage sites.

By “substantially homologous,” “substantially corresponding” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 70% homologous, preferably at least 80% homologous,more preferably at least 90% homologous, and most preferably 100%homologous to an at least 10 contiguous base region present in areference base sequence (excluding RNA and DNA equivalents). Thoseskilled in the art will readily appreciate modifications that could bemade to the hybridization assay conditions at various percentages ofhomology to permit hybridization of the oligonucleotide to the targetsequence while preventing unacceptable levels of non-specifichybridization. The degree of similarity is determined by comparing theorder of nucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of homologybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0-3 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 70% complementary, preferably at least 80%complementary, more preferably at least 90% complementary, and mostpreferably 100% complementary to an at least 10 contiguous base regionpresent in a target nucleic acid sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of complementarity to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of complementarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences which may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of complementarity between two sequencescan also be expressed in terms of the number of base mismatches presentin each set of at least 10 contiguous bases being compared, which mayrange from 0-3 base mismatches.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another base sequence byhydrogen bonding between a series of complementary bases. Complementarybase sequences may be complementary at each position in the basesequence of an oligonucleotide using standard base pairing (e.g., G:C,A:T or A:U pairing) or may contain one or more residues that are notcomplementary using standard hydrogen bonding (including abasic“nucleotides”), but in which the entire complementary base sequence iscapable of specifically hybridizing with another base sequence underappropriate hybridization conditions. Contiguous bases are preferably atleast about 70%, more preferably at least about 80%, still morepreferably at least about 90%, and most preferably about 100%complementary to a sequence to which an oligonucleotide is intended tospecifically hybridize. Appropriate hybridization conditions are wellknown to those skilled in the art, can be predicted readily based onbase sequence composition, or can be determined empirically by usingroutine testing (e.g., See Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57 particularly at §§9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

By “capture oligonucleotide” is meant at least one nucleic acidoligonucleotide that provides means for specifically joining a targetsequence and an immobilized oligonucleotide due to base pairhybridization. A capture oligonucleotide preferably includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually contiguous on the same oligonucleotide,although the capture oligonucleotide may include a targetsequence-binding region and an immobilized probe-binding region whichare present on two different oligonucleotides joined together by one ormore linkers. For example, an immobilized probe-binding region may bepresent on a first oligonucleotide, the target sequence-binding regionmay be present on a second oligonucleotide, and the two differentoligonucleotides are joined by hydrogen bonding with a linker that is athird oligonucleotide containing sequences that hybridize specificallyto the sequences of the first and second oligonucleotides.

By “immobilized probe” or “immobilized nucleic acid” is meant a nucleicacid that joins, directly or indirectly, a capture oligonucleotide to animmobilized support. An immobilized probe is an oligonucleotide joinedto a solid support that facilitates separation of bound target sequencefrom unbound material in a sample.

By “separating” or “purifying” is meant that one or more components ofthe biological sample are removed from one or more other components ofthe sample. Sample components include nucleic acids in a generallyaqueous solution phase which may also include materials such asproteins, carbohydrates, lipids and labeled probes. Preferably, theseparating or purifying step removes at least about 70%, more preferablyat least about 90% and, even more preferably, at least about 95% of theother components present in the sample.

By “RNA and DNA equivalents” or “RNA and DNA equivalent bases” is meantmolecules, such as RNA and DNA, having the same complementary base pairhybridization properties. RNA and DNA equivalents have different sugarmoieties (i.e., ribose versus deoxyribose) and may differ by thepresence of uracil in RNA and thymine in DNA. The differences betweenRNA and DNA equivalents do not contribute to differences in homologybecause the equivalents have the same degree of complementarity to aparticular sequence.

As used herein, an “in vitro amplification reaction” is anenzyme-catalyzed reaction that results in the synthesis of multiplecopies of a target nucleic acid sequence, its complement or fragmentsthereof. Examples of some useful amplification methods that can be usedfor preparing in vitro amplification reactions are given below. An“isothermal in vitro amplification reaction” is an in vitroamplification reaction that can synthesize multiple copies of a targetnucleic acid sequence, its complement or fragments thereof at a constanttemperature (i.e., without thermal cycling).

As used herein, the term “amplicons” refers to the nucleic acidamplification products of an in vitro amplification reaction. Ampliconsmay comprise DNA or RNA, depending on the nature of the in vitroamplification reaction used to produce the amplicons.

As used herein, the “target-hybridizing sequence” of a hybridizationprobe or an amplification primer refers to the base sequence of theprobe or primer which participates in a duplex structure uponhybridization to an appropriate target nucleic acid. In the case of apromoter-primer that includes a downstream sequence complementary to thetarget nucleic acid and an upstream T7 promoter sequence which is notcomplementary to the target nucleic acid, the non-complementary promotersequence of the primer would not be considered a target-hybridizingsequence. Conversely, a downstream primer sequence sufficientlycomplementary to the target nucleic acid to be able to form a duplexstructure upon hybridization to the target nucleic acid would be atarget-hybridizing sequence. If the target-hybridizing sequence of theprimer contains occasional mismatches to the target nucleic acidsequence, then it would not be fully complementary to the target nucleicacid sequence within the target nucleic acid molecule.

By “fully complementary” is meant 100% base complementarity between twopolynucleotide molecules over the length of the target-hybridizingsequence.

As used herein, monitoring amplicon production “as a function of time”refers to the process of taking periodic measurements of the amount ofamplicon present in an in vitro amplification reaction, and associatingthat measured amount with the time elapsed since initiating the in vitroamplification reaction. For example, periodic measurements can be takenat the same point of different cycles of an amplification reaction, orat periodic time intervals (such as every 20 seconds) during a reactionthat does not involve physical cycling of reaction steps.

As used herein, a “standard curve” is a representation that relates (1)a pre-amplification amount of a polynucleotide, and (2) sometime-dependent indicia of a post-amplification amount of a correspondingamplicon. For example, a standard curve can be a graph having knownnumbers of input template molecules plotted on the x-axis, and a timevalue required for the amplification reaction to achieve some level ofdetectable amplicon production plotted on the y-axis. Standard curvestypically are produced using control polynucleotide standards containingknown numbers of polynucleotide templates. Standard curves can be storedin electronic form or can be represented graphically. Thepre-amplification amount of an analyte polynucleotide in a test samplecan be determined by comparing a measured time-dependent value obtainedfor the test sample with a standard curve, as will be familiar to thosehaving an ordinary level of skill in the art.

By an “HIV-1 standard” is meant a known number of copies of an HIV-1polynucleotide, without specifying the HIV-1 genotype.

By an “HIV-1 M group standard” is meant a known number of copies of anHIV-1 M group polynucleotide.

By an “HIV-1 O group standard” is meant a known number of copies of anHIV-1 O group polynucleotide.

As used herein, the process step of “selecting” an amplification primeror hybridization probe means choosing an amplification primer orhybridization probe having certain specified features.

As used herein, two different nucleic acid targets are said to amplifywith “substantially equal efficiency” when the rates of ampliconsynthesis are substantially equal in in vitro amplification reactionsconducted using similar numbers of the two different nucleic acidtargets as templates. Practically speaking, it is not necessary toamplify all species of HIV-1 nucleic acids with identical efficienciesto achieve the benefits of the invention. Instead, it is only necessaryto use primers and a probe that will yield substantially equalamplification efficiencies. By this it is meant that, for independent invitro amplification reactions conducted using HIV-1 M group and O groupnucleic acid templates at starting levels of 1,000 copies/reaction, thedifference between the average number of starting copies/reactiondetermined for each target and the actual number of startingcopies/reaction is no greater than 1.0 log₁₀ copies/reaction, morepreferably no greater than 0.7 log₁₀ copies/reaction, and still morepreferably no greater than 0.5 log₁₀ copies/reaction.

As used herein, requiring that two primers and a probe are “selected toamplify in an in vitro amplification reaction HIV-1 M group nucleicacids and HIV-1 O group nucleic acids with substantially equalefficiencies” means that, after screening different combinations ofprimers and probes, particular combinations are chosen for thecharacteristic of amplifying HIV-1 M group and HIV-1 O group nucleicacids in in vitro amplification reactions with substantially equalefficiencies.

By “an amplification product produced by the combined activity of saidfirst and second amplification primers using as a template either HIV-1M group target nucleic acids or HIV-1 O group target nucleic acids” ismeant any amplicon synthesized using a combination of two primers, whereeach of the primers is able to use HIV-1 M group target nucleic acids orHIV-1 O group target nucleic acids, or the complements thereof, astemplates.

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions or kits or methods of the present invention. Suchcharacteristics include the ability to selectively detect HIV-1 nucleicacids in biological samples such as whole blood or plasma. Anycomponent(s), composition(s), or method step(s) that have a materialeffect on the basic and novel characteristics of the present inventionwould fall outside of this term.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the various polynucleotidesthat can be used for detecting a target region within the HIV-1 nucleicacid (represented by a thick horizontal line). Positions of thefollowing nucleic acids are shown relative to the target region:“Capture Oligonucleotide” refers to the nucleic acid used to hybridizeto and capture the target nucleic acid prior to amplification, where “T”refers to a tail sequence used to hybridize an immobilizedoligonucleotide having a complementary sequence (not shown); “Non-T7Primer” and “T7 Promoter-Primer” represent two amplification primersused for conducting TMA, where “P” indicates the promoter sequence ofthe T7 promoter-primer; and “Probe” refers to the probe used fordetecting amplified nucleic acid.

FIG. 2 is a line graph relating the amount of HIV-1 standard input intoa real-time nucleic acid amplification reaction (x-axis) and thetime-of-emergence of the measured fluorescent signal above a backgroundthreshold (y-axis). Results are shown for trials conducted using theprimer of SEQ ID NO:1 in combination with a promoter-primer having thetarget-hybridizing sequence of SEQ ID NO:13 (open squares/solid line),and using the primer of SEQ ID NO:2 in combination with apromoter-primer having the target-hybridizing sequence of SEQ ID NO:13(open triangles/dashed line).

FIG. 3 is a line graph relating the amount of HIV-1 standard input intoa real-time nucleic acid amplification reaction (x-axis) and thetime-of-emergence of the measured fluorescent signal above a backgroundthreshold (y-axis). Results represent time-dependent amplification ofHIV-1 subtype B (open triangles/solid line) and HIV-1 O group (opendiamonds/dashed line) templates using a first-strand promoter-primerthat included the target-hybridizing sequence of SEQ ID NO:13 and asecond-strand primer having the sequence of SEQ ID NO:2.

FIGS. 4A-4B are line graphs relating the amount of HIV-1 standard inputinto a real-time nucleic acid amplification reaction (x-axis) and thetime-of-emergence of the measured fluorescent signal above a backgroundthreshold (y-axis). Results represent time-dependent amplification ofHIV-1 subtype B (FIG. 4A) and HIV-1 O group (FIG. 4B) templates using afirst-strand promoter-primer that included the target-hybridizingsequence of SEQ ID NO:13 and a second-strand primer having the sequenceof SEQ ID NO:2 (open triangles/dashed lines), or a first-strandpromoter-primer that included the target-hybridizing sequence of SEQ IDNO:15 and a second-strand primer having the sequence of SEQ ID NO:2(open diamonds/solid lines).

FIGS. 5A-5B are bar graphs representing the time-of-emergence ofmeasured fluorescent signals above a background threshold (y-axis) fordifferent HIV-1 variants at 1,000 copies/reaction. FIG. 5A shows resultsfor reactions conducted using a first-strand promoter-primer thatincluded the target-hybridizing sequence of SEQ ID NO:13 and asecond-strand primer having the sequence of SEQ ID NO:2. FIG. 5B showsresults for reactions conducted using a first-strand promoter-primerthat included the target-hybridizing sequence of SEQ ID NO:15 and asecond-strand primer having the sequence of SEQ ID NO:2.

FIGS. 6A-6B show a series of bar graphs representing results fortime-dependent amplification of numerous HIV-1 variants. Amplificationprimers had the target-hybridizing sequences of SEQ ID NO:5 and SEQ IDNO:15. The molecular torch hybridization probe used in the procedure hadthe target-hybridizing sequence of SEQ ID NO:23. Results are shown foramplification reactions conducted using 1,000 copies/reaction of thedifferent HIV-1 subtypes. FIG. 6A identifies the HIV-1 nucleic acidinput into a real-time nucleic acid amplification reaction (x-axis) andthe time-of-emergence of the measured fluorescent signal above abackground threshold (y-axis). Numerical values shown above each barindicate the time-of-emergence. FIG. 6B presents the same data shown inFIG. 6A, but plots the average log₁₀ copy number on the y-axis.Numerical values shown above each bar indicate the determined averagelog₁₀ copy number.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions, methods and kits for selectivelydetecting the nucleic acids of HIV-1 in biological samples such asblood, serum, plasma or other body fluid or tissue. The probes, primersand methods of the invention can be used either in diagnosticapplications, viral-load testing applications, or for screening donatedblood and blood products or other tissues that may contain infectiousparticles.

Introduction and Overview

The present invention includes compositions (nucleic acid captureoligonucleotides, amplification oligonucleotides and probes), methodsand kits that are particularly useful for detecting HIV-1 nucleic acidsin a biological sample. To design oligonucleotide sequences appropriatefor such uses, known HIV-1 nucleic acid sequences were first compared toidentify candidate regions of the viral genome that could serve asreagents in a diagnostic assay. As a result of these comparisons, thecapture oligonucleotides, primers and probes shown schematically in FIG.1 were selected for use in an amplified assay. Portions of sequencescontaining relatively few variants between the compared sequences werechosen as starting points for designing synthetic oligonucleotidessuitable for use in capture, amplification and detection of amplifiedsequences.

Based on these analyses, the capture oligonucleotide, amplificationprimer and probe sequences presented below were designed. Those havingan ordinary level of skill in the art will appreciate that any primersequences specific for an HIV-1 target, with or without a T7 promotersequence, may be used as primers in the various primer-based in vitroamplification methods described below. It is also contemplated thatoligonucleotides having the sequences disclosed herein could servealternative functions in assays for detecting HIV-1 nucleic acids. Forexample, the capture oligonucleotides disclosed herein could serve ashybridization probes, the hybridization probes disclosed herein could beused as amplification primers, and the amplification primers disclosedherein could be used as hybridization probes in alternative detectionassays.

Useful Amplification Methods

Amplification methods useful in connection with the present inventioninclude: Transcription Mediated Amplification (TMA), Nucleic AcidSequence-Based Amplification (NASBA), the Polymerase Chain Reaction(PCR), Strand Displacement Amplification (SDA), and amplificationmethods using self-replicating polynucleotide molecules and replicationenzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying outthese various amplification techniques respectively can be found in U.S.Pat. No. 5,399,491, U.S. Pat. No. 5,554,517, U.S. Pat. No. 4,965,188,U.S. Pat. No. 5,455,166, U.S. Pat. No. 5,472,840 and Lizardi et al.,BioTechnology 6:1197 (1988). The disclosures of these documents whichdescribe how to perform nucleic acid amplification reactions are herebyincorporated by reference.

In a highly preferred embodiment of the invention, HIV-1 nucleic acidsequences are amplified using a TMA protocol. According to thisprotocol, the reverse transcriptase which provides the DNA polymeraseactivity also possesses an endogenous RNase H activity. One of theprimers used in this procedure contains a promoter sequence positionedupstream of a sequence that is complementary to one strand of a targetnucleic acid that is to be amplified. In the first step of theamplification, a promoter-primer hybridizes to the HIV-1 target RNA at adefined site. Reverse transcriptase creates a complementary DNA copy ofthe target RNA by extension from the 3′ end of the promoter-primer.Following interaction of an opposite strand primer with the newlysynthesized DNA strand, a second strand of DNA is synthesized from theend of the primer by reverse transcriptase, thereby creating adouble-stranded DNA molecule. RNA polymerase recognizes the promotersequence in this double-stranded DNA template and initiatestranscription. Each of the newly synthesized RNA amplicons re-enters theTMA process and serves as a template for a new round of replication,thereby leading to an exponential expansion of the RNA amplicon. Sinceeach of the DNA templates can make 100-1000 copies of RNA amplicon, thisexpansion can result in the production of 10 billion amplicons in lessthan one hour. The entire process is autocatalytic and is performed at aconstant temperature.

Structural Features of Primers

As indicated above, a “primer” refers to an optionally modifiedoligonucleotide which is capable of participating in a nucleic acidamplification reaction. Preferred primers are capable of hybridizing toa template nucleic acid and which has a 3′ end that can be extended by aDNA polymerase activity. The 5′ region of the primer may benon-complementary to the target nucleic acid. If the 5′non-complementary region includes a promoter sequence, it is referred toas a “promoter-primer.” Those skilled in the art will appreciate thatany oligonucleotide that can function as a primer (i.e., anoligonucleotide that hybridizes specifically to a target sequence andhas a 3′ end capable of extension by a DNA polymerase activity) can bemodified to include a 5′ promoter sequence, and thus could function as apromoter-primer. Similarly, any promoter-primer can be modified byremoval of, or synthesis without, a promoter sequence and still functionas a primer.

Nucleotide base moieties of primers may be modified (e.g., by theaddition of propyne groups), as long as the modified base moiety retainsthe ability to form a non-covalent association with G, A, C, T or U, andas long as an oligonucleotide comprising at least one modifiednucleotide base moiety or analog is not sterically prevented fromhybridizing with a single-stranded nucleic acid. As indicated below inconnection with the chemical composition of useful probes, thenitrogenous bases of primers in accordance with the invention may beconventional bases (A, G, C, T, U), known analogs thereof (e.g., inosineor “I” having hypoxanthine as its base moiety; see The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), knownderivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). Common sugar moieties thatcomprise the primer backbone include ribose and deoxyribose, although2′-O-methyl ribose (OMe), halogenated sugars, and other modified sugarmoieties may also be used. Usually, the linking group of the primerbackbone is a phosphorus-containing moiety, most commonly aphosphodiester linkage, although other linkages, such as, for example,phosphorothioates, methylphosphonates, and non-phosphorus-containinglinkages such as peptide-like linkages found in “peptide nucleic acids”(PNA) also are intended for use in the assay disclosed herein.

Useful Probe Labeling Systems and Detectable Moieties

Essentially any labeling and detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the present invention. Included among the collection ofuseful labels are radiolabels, enzymes, haptens, linkedoligonucleotides, chemiluminescent molecules, fluorescent moieties(either alone or in combination with “quencher” moieties), andredox-active moieties that are amenable to electronic detection methods.Preferred chemiluminescent molecules include acridinium esters of thetype disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction. Thedisclosures contained in these patent documents are hereby incorporatedby reference. Preferred electronic labeling and detection approaches aredisclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, and the publishedinternational patent application WO 98/57158, the disclosures of whichare hereby incorporated by reference. Redox active moieties useful aslabels in the present invention include transition metals such as Cd,Mg, Cu, Co, Pd, Zn, Fe and Ru.

Particularly preferred detectable labels for probes in accordance withthe present invention are detectable in homogeneous assay systems (i.e.,where, in a mixture, bound labeled probe exhibits a detectable change,such as stability or differential degradation, compared to unboundlabeled probe). Examples of homogeneously detectable labels includefluorescent labels, electronically detectable labels, andchemiluminescent compounds (e.g., as described by Woodhead et al., inU.S. Pat. No. 5,656,207; by Nelson et al., in U.S. Pat. No. 5,658,737;or by Arnold et al., in U.S. Pat. No. 5,639,604).

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “Molecular Beacons” comprise nucleic acidmolecules having a target complementary sequence, an affinity pair (ornucleic acid arms) holding the probe in a closed conformation in theabsence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe target nucleic acid and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare fully described in U.S. Pat. No. 5,925,517, the disclosure of whichis hereby incorporated by reference. Molecular beacons useful fordetecting HIV-1 specific nucleic acid sequences may be created byappending to either end of one of the probe sequences disclosed herein,a first nucleic acid arm comprising a fluorophore and a second nucleicacid arm comprising a quencher moiety. In this configuration, the HIV-1specific probe sequence disclosed herein serves as thetarget-complementary “loop” portion of the resulting molecular beacon,while the self-complementary “arms” of the probe represent the “stem”portion of the probe.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the invention is a structure commonlyreferred to as a “Molecular Torch.” These self-reporting probes aredesigned to include distinct regions of self-complementarity (coined“the target binding domain” and “the target closing domain”) which areconnected by a joining region and which hybridize to one another underpredetermined hybridization assay conditions. When exposed to anappropriate target or denaturing conditions, the two complementaryregions (which may be fully or partially complementary) of the moleculartorch melt, leaving the target binding domain available forhybridization to a target sequence when the predetermined hybridizationassay conditions are restored. Molecular torches are designed so thatthe target binding domain favors hybridization to the target sequenceover the target closing domain. The target binding domain and the targetclosing domain of a molecular torch include interacting labels (e.g.,fluorescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized as opposed to when themolecular torch is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label associatedtherewith. Molecular torches are fully described in U.S. Pat. No.6,361,945, the disclosure of which is hereby incorporated by reference.

Molecular torches and molecular beacons preferably are labeled with aninteractive pair of detectable labels. Examples of detectable labelsthat are preferred as members of an interactive pair of labels interactwith each other by FRET or non-FRET energy transfer mechanisms.Fluorescence resonance energy transfer (FRET) involves the radiationlesstransmission of energy quanta from the site of absorption to the site ofits utilization in the molecule, or system of molecules, by resonanceinteraction between chromophores, over distances considerably greaterthan interatomic distances, without conversion to thermal energy, andwithout the donor and acceptor coming into kinetic collision. The“donor” is the moiety that initially absorbs the energy, and the“acceptor” is the moiety to which the energy is subsequentlytransferred. In addition to FRET, there are at least three other“non-FRET” energy transfer processes by which excitation energy can betransferred from a donor to an acceptor molecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Highly preferred label moieties for the invented molecular torches andmolecular beacons include a fluorophore and a second moiety havingfluorescence quenching properties (i.e., a “quencher”). In thisembodiment, the characteristic signal is likely fluorescence of aparticular wavelength, but alternatively could be a visible lightsignal. When fluorescence is involved, changes in emission arepreferably due to FRET, or to radiative energy transfer or non-FRETmodes. When a molecular beacon having a pair of interactive labels inthe closed state is stimulated by an appropriate frequency of light, afluorescent signal is generated at a first level, which may be very low.When this same probe is in the open state and is stimulated by anappropriate frequency of light, the fluorophore and the quenchermoieties are sufficiently separated from each other that energy transferbetween them is substantially precluded. Under that condition, thequencher moiety is unable to quench the fluorescence from thefluorophore moiety. If the fluorophore is stimulated by light energy ofan appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used in connectionwith the invention, making no attempt to distinguish FRET from non-FRETpairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluororescein,EDANS/DABCYL, coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPYFL, fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL,eosine/DABCYL, erythrosine/DABCYL, tetramethylrhodamine/DABCYL, TexasRed/DABCYL, CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2 and fluorescein/QSY7 dye.Those having an ordinary level of skill in the art will understand thatwhen donor and acceptor dyes are different, energy transfer can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor speciesare the same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BLACKHOLE QUENCHER moieties which are available from Biosearch Technologies,Inc., (Novato, Calif.).

Synthetic techniques and methods of bonding labels to nucleic acids anddetecting labels are well known in the art (e.g., see Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., U.S. Pat. No. 4,581,333), and Becker etal., European Patent App. No. 0 747 706.

Chemical Composition of Probes

Probes in accordance with the invention comprise polynucleotides orpolynucleotide analogs and optionally may carry a detectable labelcovalently bonded thereto. Nucleosides or nucleoside analogs of theprobe comprise nitrogenous heterocyclic bases, or base analogs, wherethe nucleosides are linked together, for example by phosphohdiesterbonds to form a polynucleotide. Accordingly, a probe may compriseconventional ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA),but also may comprise chemical analogs of these molecules. The“backbone” of a probe may be made up of a variety of linkages known inthe art, including one or more sugar-phosphodiester linkages,peptide-nucleic acid bonds (sometimes referred to as “peptide nucleicacids” as described by Hyldig-Nielsen et al., PCT Int'l Pub. No. WO95/32305), phosphorothioate linkages, methylphosphonate linkages orcombinations thereof. Sugar moieties of the probe may be either riboseor deoxyribose, or similar compounds having known substitutions, suchas, for example, 2′-O-methyl ribose and 2′ halide substitutions (e.g.,2′-F). The nitrogenous bases may be conventional bases (A, G, C, T, U),known analogs thereof (e.g., inosine or “I”; see The Biochemistry of theNucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), knownderivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). A probe may comprise onlyconventional sugars, bases and linkages found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs).

While oligonucleotide probes of different lengths and base compositionmay be used for detecting HIV-1 nucleic acids, preferred probes in thisinvention have lengths of up to 100 nucleotides, and more preferablyhave lengths of up to 60 nucleotides. Preferred length ranges for theinvented oligonucleotides are from 10 to 100 bases in length, or morepreferably between 15 and 50 bases in length, or still more preferablybetween 15 and 30 bases in length.

However, the specific probe sequences described below also may beprovided in a nucleic acid cloning vector or transcript or other longernucleic acid and still can be used for detecting HIV-1 nucleic acids.

Selection of Amplification Primers and Detection Probes Specific forHIV-1

Useful guidelines for designing amplification primers and probes withdesired characteristics are described herein. The optimal sites foramplifying and probing HIV-1 nucleic acids contain two, and preferablythree, conserved regions each greater than about 15 bases in length,within about 200 bases of contiguous sequence. The degree ofamplification observed with a set of primers or promoter-primers dependson several factors, including the ability of the oligonucleotides tohybridize to their complementary sequences and their ability to beextended enzymatically. Because the extent and specificity ofhybridization reactions are affected by a number of factors,manipulation of those factors will determine the exact sensitivity andspecificity of a particular oligonucleotide, whether perfectlycomplementary to its target or not. The effects of varying assayconditions are known to those skilled in the art, and are described byHogan et al., in U.S. Pat. No. 5,840,488, the disclosure of which ishereby incorporated by reference.

The length of the target nucleic acid sequence and, accordingly, thelength of the primer sequence or probe sequence can be important. Insome cases, there may be several sequences from a particular targetregion, varying in location and length, which will yield primers orprobes having the desired hybridization characteristics. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencewill normally primarily determine hybrid stability.

Amplification primers and probes should be positioned to minimize thestability of the oligonucleotide:nontarget (i.e., nucleic acid withsimilar sequence to target nucleic acid) nucleic acid hybrid. It ispreferred that the amplification primers and detection probes are ableto distinguish between target and non-target sequences. In designingprimers and probes, the differences in these Tm values should be aslarge as possible (e.g., at least 2° C. and preferably 5° C.).

The degree of non-specific extension (primer-dimer or non-targetcopying) can also affect amplification efficiency. For this reason,primers are selected to have low self- or cross-complementarity,particularly at the 3′ ends of the sequence. Long homopolymer tracts andhigh GC content are avoided to reduce spurious primer extension.Commercially available computer software can aid in this aspect of thedesign. Available computer programs include MacDNASIS™ 2.0 (HitachiSoftware Engineering American Ltd.) and OLIGO ver. 6.6 (MolecularBiology Insights; Cascade, Colo.).

Those having an ordinary level of skill in the art will appreciate thathybridization involves the association of two single strands ofcomplementary nucleic acid to form a hydrogen bonded double strand. Itis implicit that if one of the two strands is wholly or partiallyinvolved in a hybrid, then that strand will be less able to participatein formation of a new hybrid. By designing primers and probes so thatsubstantial portions of the sequences of interest are single stranded,the rate and extent of hybridization may be greatly increased. If thetarget is an integrated genomic sequence, then it will naturally occurin a double stranded form (as is the case with the product of thepolymerase chain reaction). These double-stranded targets are naturallyinhibitory to hybridization with a probe and require denaturation priorto the hybridization step.

The rate at which a polynucleotide hybridizes to its target is a measureof the thermal stability of the target secondary structure in the targetbinding region. The standard measurement of hybridization rate is theC₀t_(1/2) which is measured as moles of nucleotide per liter multipliedby seconds. Thus, it is the concentration of probe multiplied by thetime at which 50% of maximal hybridization occurs at that concentration.This value is determined by hybridizing various amounts ofpolynucleotide to a constant amount of target for a fixed time. TheC₀t_(1/2) is found graphically by standard procedures familiar to thosehaving an ordinary level of skill in the art.

Preferred Amplification Primers

Primers useful for conducting amplification reactions can have differentlengths to accommodate the presence of extraneous sequences that do notparticipate in target binding, and that may not substantially affectamplification or detection procedures. For example, promoter-primersuseful for performing amplification reactions in accordance with theinvention have at least a minimal sequence that hybridizes to the HIV-1target nucleic acid, and a promoter sequence positioned upstream of thatminimal sequence. However, insertion of sequences between the targetbinding sequence and the promoter sequence could change the length ofthe primer without compromising its utility in the amplificationreaction. Additionally, the lengths of the amplification primers anddetection probes are matters of choice as long as the sequences of theseoligonucleotides conform to the minimal essential requirements forhybridizing the desired complementary sequence.

Tables 1 and 2 present specific examples of oligonucleotide sequencesthat were used as primers for amplifying HIV-1 nucleic acids in the polregion. Table 1 presents the sequences of primers that werecomplementary to HIV-1 sequences on one strand of nucleic acid. Table 2presents the sequences of both the HIV-1 target-complementary primersand the full sequences for promoter-primers that were used duringdevelopment of the invention. Notably, the oligonucleotide sequences inTable 1 and Table 2 are complementary to opposite strands of the HIV-1nucleic acid.

TABLE 1 Polynucleotide Sequences of Amplification Primers SequenceSEQ ID NO: ACAGCAGTACAAATGGCAG 1 CCACAATTTTAAAAGAAAAGGG 2CCACAATTTTAAGAGAAAAGGG 3 CCACAATTTTAGAAGAAAAGGG 4 CCACAATTTTGAAAGAAAAGGG5 CCACAATTTTAAAGGAAAAGGG 6 CCACAATTTGAAAAGAAAAGGG 7CCACAGTTTTAAAAGAAAAGGG 8 CCACAATTTTGAAAGAAAAGGGG 9CCACAATATTAAAAGAAAAGGG 10 CCACAATTTTAAAAGAGAAGGGGGGATTGG 11CCACAATTTTAAAAGGAAAGGGGGGATTGG 12

Table 2 presents HIV-1 target-complementary oligonucleotide sequencesand the corresponding promoter-primer sequences that were used foramplifying HIV-1 nucleic acid sequences in the HIV-1 pol region. Asindicated above, promoter-primers that are to be used for practicing theinvention include sequences complementary to an HIV-1 target sequence attheir 3′ ends, and a T7 promoter sequence (presented in lowercase) attheir 5′ ends.

TABLE 2 Polynucleotide Sequences of Amplification Primers SequenceSEQ ID NO: AGTTTGTATGTCTGTTGCTATTATGTCTA 13AGTTTGTGTGTCTGTTGCTGTTATGTCTA 14 AGTTTGTATGTCTGATGCTATTATGTCTA 15AGTTTGTATGTCTGGTGCTATTATGTCTA 16 5′-aatttaatacgactcactatagggag- 17AGTTTGTATGTCTGTTGCTATTATGTCTA-3′ 5′-aatttaatacgactcactatagggag- 18AGTTTGTGTGTCTGTTGCTGTTATGTCTA-3′ 5′-aatttaatacgactcactatagggag- 19AGTTTGTATGTCTGATGCTATTATGTCTA-3′ 5′-aatttaatacgactcactatagggag- 20AGTTTGTATGTCTGGTGCTATTATGTCTA-3′

Preferred sets of primers for amplifying HIV-1 sequences in the polregion include a first primer that hybridizes an HIV-1 target sequence(such as one of the primers listed in Table 2) and a second primercomplementary to the sequence of an extension product of the firstprimer (such as one of the primer sequences listed in Table 1). In ahighly preferred embodiment, the first primer is a promoter-primer thatincludes a T7 promoter sequence at its 5′ end.

Preferred Detection Probes

Another aspect of the invention relates to hybridization probes fordetecting HIV-1 nucleic acids. Methods for amplifying a target nucleicacid sequence present in the nucleic acid of HIV-1 can include anoptional further step for detecting amplicons. This method includes astep for contacting a test sample with a hybridization assay probe thatpreferentially hybridizes to the target nucleic acid sequence, or thecomplement thereof, thereby forming a probe:target duplex that is stablefor detection. Next there is a step for determining whether the hybridis present in the test sample as an indication of the presence orabsence of HIV-1 nucleic acids in the test sample. This may involvedetecting the probe:target duplex, and preferably involves homogeneousassay systems.

Hybridization assay probes useful for detecting HIV-1 nucleic acidsequences include a sequence of bases substantially complementary to anHIV-1 target nucleic acid sequence. Thus, probes of the inventionpreferably hybridize one strand of an HIV-1 target nucleic acidsequence, or the complement thereof. These probes may optionally haveadditional bases outside of the targeted nucleic acid region which mayor may not be complementary to the HIV-1 nucleic acid.

Certain highly preferred probes are able to hybridize to HIV-1 targetnucleic acids under conditions suitable for performing a nucleic acidamplification reaction, such as those described herein. Examples ofparticularly preferred probes useful in connection with this aspect ofthe invention include molecular beacons and molecular torches.

Other preferred probes are sufficiently homologous to the target nucleicacid to hybridize under stringent hybridization conditions correspondingto about 60° C. when the salt concentration is in the range of 0.6-0.9M. Preferred salts include lithium chloride, but other salts such assodium chloride and sodium citrate also can be used in the hybridizationsolution. Example high stringency hybridization conditions arealternatively provided by 0.48 M sodium phosphate buffer, 0.1% sodiumdodecyl sulfate, and 1 mM each of EDTA and EGTA, or by 0.6 M LiCl, 1%lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTAand EGTA.

Probes in accordance with the invention have sequences complementary to,or corresponding to a portion of the HIV-1 genome. Certain probes thatare preferred for detecting HIV-1 nucleic acid sequences have a probesequence, which includes the target-complementary sequence of basestogether with any base sequences that are not complementary to thenucleic acid that is to be detected, in the length range of from 10-100nucleotides. Certain specific probes that are preferred for detectingHIV-1 nucleic acid sequences have target-complementary sequences in thelength range of from 10-50, from 10-20, or from 10-15 nucleotides. Ofcourse, these target-complementary sequences may be linear sequences, ormay be contained in the structure of a molecular beacon, molecular torchor other construct having one or more optional nucleic acid sequencesthat are non-complementary to the HIV-1 target sequence that is to bedetected. As indicated above, probes may be made of DNA, RNA, acombination of DNA and RNA, a nucleic acid analog, or contain one ormore modified nucleosides (e.g., a ribonucleoside having a 2′-O-methylsubstitution to the ribofuranosyl moiety).

Certain highly preferred probes include a detectable label. In oneembodiment, the detectable label is a fluorescent label which may,optionally, be used in combination with a quencher moiety. In otherembodiments, the label is an acridinium ester joined to the probe bymeans of a non-nucleotide linker. For example, detection probes can belabeled with chemiluminescent acridinium ester compounds that areattached via a linker substantially as described in U.S. Pat. No.5,585,481; and in U.S. Pat. No. 5,639,604, particularly as described atcolumn 10, line 6 to column 11, line 3, and in Example 8. Thedisclosures contained in these patent documents are hereby incorporatedby reference. Of course, highly preferred probes for use intime-dependent amplicon detection include molecular beacons andmolecular torches.

Table 3 presents the target-complementary base sequences, and fullsequences of some of the hybridization probes that were used fordetecting HIV-1 amplicons. Since alternative probes for detecting HIV-1nucleic acid sequences can hybridize to the opposite-sense strand ofHIV-1, the present invention also includes oligonucleotides that arecomplementary to the sequences presented in the table. Thetarget-hybridizing sequence of SEQ ID NO:21 was incorporated into themolecular beacon having the sequence of SEQ ID NO:22. Thetarget-hybridizing sequence of SEQ ID NO:23 was incorporated into themolecular torch having the sequence of SEQ ID NO:24. Both the molecularbeacon and the molecular torch appearing in Table 3 were labeled with afluorescein moiety at its 5′ end, and with a DABCYL quencher moiety atits 3′ end.

TABLE 3 Polynucleotide Sequences of  HIV-1 Detection Probes SequenceSEQ ID NO: UGGIGGGUACAGUGC 21 CCGUGGIGGGUACAGUGCCACGG3′ 22GGIGGGUACAGUGC 23 CGGIGGGUACAGUGC (C9) CCCCG 24

As indicated above, any number of different backbone structures can beused as a scaffold for the nucleobase sequences of the inventedhybridization probes. In certain highly preferred embodiments, the probesequence used for detecting HIV-1 amplicons includes a methoxy backbone,or at least one methoxy linkage in the nucleic acid backbone.

Selection and Use of Capture Oligonucleotides

Preferred capture oligonucleotides include a first sequence that iscomplementary to an HIV-1 sequence (i.e., an “HIV-1 target sequence”)covalently attached to a second sequence (i.e., a “tail” sequence) thatserves as a target for immobilization on a solid support. Any backboneto link the base sequence of a capture oligonucleotide may be used. Incertain preferred embodiments the capture oligonucleotide includes atleast one methoxy linkage in the backbone. The tail sequence, which ispreferably at the 3′ end of a capture oligonucleotide, is used tohybridize to a complementary base sequence to provide a means forcapturing the hybridized target HIV-1 nucleic acid in preference toother components in the biological sample.

Although any base sequence that hybridizes to a complementary basesequence may be used in the tail sequence, it is preferred that thehybridizing sequence span a length of about 5-50 nucleotide residues.Particularly preferred tail sequences are substantially homopolymeric,containing about 10 to about 40 nucleotide residues, or more preferablyabout 14 to about 30 residues. A capture oligonucleotide according tothe present invention may include a first sequence that specificallybinds an HIV-1 target polynucleotide, and a second sequence thatspecifically binds an oligo(dT) stretch immobilized to a solid support.

Using the components illustrated in FIG. 1, one assay for detectingHIV-1 sequences in a biological sample includes the steps of capturingthe target nucleic acid using the capture oligonucleotide, amplifyingthe captured target region using at least two primers, and detecting theamplified nucleic acid by first hybridizing the labeled probe to asequence contained in the amplified nucleic acid and then detecting asignal resulting from the bound labeled probe.

The capturing step preferably uses a capture oligonucleotide where,under hybridizing conditions, one portion of the capture oligonucleotidespecifically hybridizes to a sequence in the target nucleic acid and atail portion serves as one component of a binding pair, such as a ligand(e.g., a biotin-avidin binding pair) that allows the target region to beseparated from other components of the sample. Preferably, the tailportion of the capture oligonucleotide is a sequence that hybridizes toa complementary sequence immobilized to a solid support particle.Preferably, first, the capture oligonucleotide and the target nucleicacid are in solution to take advantage of solution phase hybridizationkinetics. Hybridization produces a capture oligonucleotide:targetnucleic acid complex which can bind an immobilized probe throughhybridization of the tail portion of the capture oligonucleotide with acomplementary immobilized sequence. Thus, a complex comprising a targetnucleic acid, capture oligonucleotide and immobilized probe is formedunder hybridization conditions. Preferably, the immobilized probe is arepetitious sequence, and more preferably a homopolymeric sequence(e.g., poly-A, poly-T, poly-C or poly-G), which is complementary to thetail sequence and attached to a solid support. For example, if the tailportion of the capture oligonucleotide contains a poly-A sequence, thenthe immobilized probe would contain a poly-T sequence, although anycombination of complementary sequences may be used. The captureoligonucleotide may also contain “spacer” residues, which are one ormore bases located between the base sequence that hybridizes to thetarget and the base sequence of the tail that hybridizes to theimmobilized probe. Any solid support may be used for binding the targetnucleic acid:capture oligonucleotide complex. Useful supports may beeither matrices or particles free in solution (e.g., nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and, preferably, magnetically attractable particles).Methods of attaching an immobilized probe to the solid support are wellknown. The support is preferably a particle which can be retrieved fromsolution using standard methods (e.g., centrifugation, magneticattraction of magnetic particles, and the like). Preferred supports areparamagnetic monodisperse particles (i.e., uniform in size±about 5%).

Retrieving the target nucleic acid:capture oligonucleotide:immobilizedprobe complex effectively concentrates the target nucleic acid (relativeto its concentration in the biological sample) and purifies the targetnucleic acid from amplification inhibitors which may be present in thebiological sample. The captured target nucleic acid may be washed one ormore times, further purifying the target, for example, by resuspendingthe particles with the attached target nucleic acid:captureoligonucleotide:immobilized probe complex in a washing solution and thenretrieving the particles with the attached complex from the washingsolution as described above. In a preferred embodiment, the capturingstep takes place by sequentially hybridizing the capture oligonucleotidewith the target nucleic acid and then adjusting the hybridizationconditions to allow hybridization of the tail portion of the captureoligonucleotide with an immobilized complementary sequence (e.g., asdescribed in PCT No. WO 98/50583). After the capturing step and anyoptional washing steps have been completed, the target nucleic acid canthen be amplified. To limit the number of handling steps, the targetnucleic acid optionally can be amplified without releasing it from thecapture oligonucleotide.

Useful capture oligonucleotides may contain mismatches to theabove-indicated sequences, as long as the mismatched sequences hybridizeto the HIV-1 nucleic acid containing the sequence that is to beamplified. Each capture oligonucleotide described herein included one ofthe HIV-1 sequences presented in Table 4 linked to a poly-(dA) tail atits 3′ end. All of the capture oligonucleotides also included threeoptional thymidine nucleotides interposed between the HIV-1complementary sequence and the poly-(dA) tail. The presence of thesethymidine nucleotides is not believed to be essential for success of thecapture procedure. The three thymidine nucleotides and the poly-(dA)tail were synthesized using DNA precursors, while the HIV-1complementary portions of the oligonucleotides were synthesized using2′-OMe nucleotide analogs.

TABLE 4 HIV-1 Complementary Portions of Capture OligonucleotidesSequence SEQ ID NO: GCUGGAAUAACUUCUGCUUCUAU 25 GCUGGAAUAGCUUCUGCUUCUAU26 UCUGCUGUCCCUGUAAUAAACCCG 27 UCUGCUGUCCCUGUGAUAAACCCG 28

Preferred Methods for Amplifying and Detecting HIV-1 PolynucleotideSequences

Preferred methods of the present invention are described and illustratedby the Examples presented below. FIG. 1 schematically illustrates onesystem that may be used for detecting a target region of the HIV-1genome (shown by a thick solid horizontal line). This system includesfour oligonucleotides (shown by the shorter solid lines): one captureoligonucleotide that includes a sequence that hybridizes specifically toan HIV-1 sequence in the target region and a tail (“T”) that hybridizesto a complementary sequence immobilized on a solid support to capturethe target region present in a biological sample; one T7 promoter-primerwhich includes a sequence that hybridizes specifically to an HIV-1sequence in the target region and a T7 promoter sequence (“P”) which,when double-stranded, serves as a functional promoter for T7 RNApolymerase; one non-T7 primer which includes a sequence that hybridizesspecifically to a first strand cDNA made from the target region sequenceusing the T7 promoter-primer; and one labeled probe which includes asequence that hybridizes specifically to a portion of the target regionthat is amplified using the two primers.

As indicated above, amplifying the captured target region using the twoprimers can be accomplished by any of a variety of known nucleic acidamplification reactions that will be familiar to those having anordinary level of skill in the art. In a preferred embodiment, atranscription-associated amplification reaction, such as TMA, isemployed. In such an embodiment, many strands of nucleic acid areproduced from a single copy of target nucleic acid, thus permittingdetection of the target by detecting probes that are bound to theamplified sequences. Preferably, transcription-associated amplificationuses two types of primers (one being referred to as a promoter-primerbecause it contains a promoter sequence, labeled “P” in FIG. 1, for anRNA polymerase) two enzymes (a reverse transcriptase and an RNApolymerase), and substrates (deoxyribonucleoside triphosphates,ribonucleoside triphosphates) with appropriate salts and buffers insolution to produce multiple RNA transcripts from a nucleic acidtemplate.

Referring to FIG. 1, during transcription-mediated amplification, thecaptured target nucleic acid is hybridized to a first primer shown as aT7 promoter-primer. Using reverse transcriptase, a complementary DNAstrand is synthesized from the T7 promoter-primer using the target DNAas a template. A second primer, shown as a non-T7 primer, hybridizes tothe newly synthesized DNA strand and is extended by the action of areverse transcriptase to form a DNA duplex, thereby forming adouble-stranded T7 promoter region. T7 RNA polymerase then generatesmultiple RNA transcripts by using this functional T7 promoter. Theautocatalytic mechanism of TMA employs repetitive hybridization andpolymerization steps following a cDNA synthesis step using the RNAtranscripts as templates to produce additional transcripts, therebyamplifying target region-specific nucleic acid sequences.

The detecting step uses at least one detection probe that bindsspecifically to the amplified RNA transcripts or amplicons describedabove. Preferably, the detection probe is labeled with a label that canbe detected using a homogeneous detection system. For example, thelabeled probe can be labeled with an acridinium ester compound fromwhich a chemiluminescent signal may be produced and detected, asdescribed above. Alternatively, the labeled probe may comprise afluorophore or a combination of fluorophore and quencher moieties.Molecular beacons and molecular torches are alternative embodiments ofsuch labeled probes that may be used in homogeneous detection systems.

Use of a Standard Curve—Quantifying Pre-Amplification Amounts of AnalytePolynucleotide

In general, the invented methods can involve the step of consulting astandard curve that relates pre-amplification amounts of analytepolynucleotide and post-amplification amounts of analyte amplicon.

Since real-time amplification reactions advantageously featurequantitative relationships between the number of analyte polynucleotidesinput into the reaction and the number of analyte amplicons synthesizedas a function of time, the number of analyte polynucleotides present ina test sample can be determined using a standard curve. For example, aplurality of amplification reactions containing known amounts of apolynucleotide standard can be run in parallel with an amplificationreaction prepared using a test sample containing an unknown number ofanalyte polynucleotides. Alternatively, a standard curve can be preparedin advance so that it is unnecessary to prepare a curve each time ananalytical procedure is carried out. Such a curve prepared in advancecan even be stored electronically in a memory device of a testinginstrument. A standard curve having pre-amplification amounts of thepolynucleotide standard on a first axis and some indicia of the timerequired to effect a certain level of nucleic acid amplification (suchas a time-of-emergence above a background signal) on a second axis isthen prepared. The post-amplification amount of analyte ampliconmeasured for the test reaction is then located on the post-amplificationaxis of the standard curve. The corresponding value on the other axis ofthe curve represents the pre-amplification amount of analytepolynucleotide that was present in the test reaction. Thus, determiningthe number of molecules of analyte polynucleotide present in the testsample is accomplished by consulting the standard curve, or moreparticularly by comparing the quantitative results obtained for the testsample with the standard curve, a procedure that will be familiar tothose having an ordinary level of skill in the art.

The procedures described herein can easily be used to quantify analytepolynucleotides present in a test sample. Indeed, if a plurality ofstandard control amplification reactions are initiated using knownnumbers of an analyte polynucleotide standard, and if a test reactionthat includes an unknown number of analyte polynucleotide molecules iscarried out, then it becomes possible after measuring the time requiredto effect a certain level of amplification in each reaction to determinethe number of analyte polynucleotide molecules that must have beenpresent in the test sample. The relationship between the number ofanalyte polynucleotide molecules input into standard amplificationreaction and the time required to effect a certain level ofamplification is conveniently established using a graph. Determining thenumber of analyte polynucleotide molecules present in a test sample issimply a matter of determining from the standard graph the number ofanalyte polynucleotide molecules that correspond to a measured analyteamplicon signal strength. This illustrates how analyte polynucleotidestandards can be used in connection with polynucleotide amplificationreactions to quantify pre-amplification amounts of analytepolynucleotide contained in test samples.

Kits for Detecting HIV-1 Nucleic Acids

The present invention also embraces kits for performing polynucleotideamplification reactions using viral nucleic acid templates. Certainpreferred kits will contain a hybridization assay probe that includes atarget-complementary sequence of bases, and optionally primers or otherancillary oligonucleotides for amplifying the target that is to bedetected. Other preferred kits will contain a pair of oligonucleotideprimers that may be used for amplifying target nucleic acids in an invitro amplification reaction. Exemplary kits include first and secondamplification oligonucleotides that are complementary to oppositestrands of an HIV-1 nucleic acid sequence that is to be amplified. Thekits may further contain one or more oligonucleotide detection probes.Still other kits in accordance with the invention may additionallyinclude capture oligonucleotides for purifying HIV-1 template nucleicacids away from other species prior to amplification.

The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting Examples. TheseExamples describe the development of quantitative nucleic acidamplification assays characterized by substantially linear relationshipsbetween the time required to yield a positive amplification signal andthe initial amount of HIV-1 template nucleic acid included in thereaction. The invented assays are further characterized by high levelsof precision in the quantitation of HIV-1 targets at low copy numbers,and by accurate detection of different HIV-1 subtypes, including M groupand O group variants.

Oligonucleotide primers disclosed in published international applicationWO 2003106714, together with a molecular beacon, served as a startingpoint for the development of the invented assay. As indicated by theevidence presented in Example 1, modifying the initial primer set bysubstituting one of the primers dramatically improved the quantitativecapacity of the assay by increasing the detectability of low levels ofthe HIV-1 template. In all cases, positive amplification was indicatedby the time-dependent appearance of a fluorescent signal in homogeneousassays.

Analysis of the experimental data was performed using acomputer-implemented algorithm to establish a substantially linearrelationship between the number of HIV-1 template copies included in anamplification reaction and the time at which the fluorescent signalexceeded a background value (i.e., “time-of-emergence” abovebackground). Essentially identical analyses were conducted for all ofthe time-dependent assays disclosed herein.

As confirmed by the results presented below, similar procedures can beused for quantifying analyte target amounts present in a test sample.More specifically, when known amounts of an analyte polynucleotide areused as calibration standards, it is possible to determine the amount ofanalyte present in a test sample by comparing the time-dependentappearance of a fluorescent signal measured for the test sample with astandard curve.

Example 1 describes procedures wherein a molecular beacon probe labeledwith an interactive fluorophore/quencher pair was used for monitoringtime-dependent amplicon production in nucleic acid amplificationreactions. Although the molecular beacons described in this Examplehybridized to only one strand of the amplified nucleic acid product,probe sequences complementary to the HIV-1 nucleic acid on the oppositestrand also fall within the scope of the invention. Results from theseprocedures indicated that the choice of oligonucleotide primersprofoundly affected the quantitative capacity of the assay.

Example 1 Time-Dependent Monitoring of HIV-1 M Group, Subtype B AmpliconProduction

An in vitro synthesized transcript of known concentration included thesequence

(SEQ ID NO: 29)GGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGAT,and served as the source of HIV-1 subtype B template sequences inamplification reactions that employed paired sets of primers. This invitro transcript contained portions of the HIV-1 genome that includedsequences substantially corresponding to, or substantially complementaryto, each of the primers used in the procedure. Nucleic acidamplification reactions were performed using a TMA protocol, and werecarried out essentially as described by Kacian et al., in U.S. Pat. No.5,399,491, the disclosure of this U.S. patent having been incorporatedby reference hereinabove. Promoter-primers used in the TMA reactionsincluded a T7 promoter sequence AATTTAATACGACTCACTATAGGGAG (SEQ IDNO:30) appended upstream of an HIV-1 complementary sequence. Thesequence of the T7 promoter is absent from the HIV-1 analytepolynucleotide, and so was not complementary to the HIV-1 template.Amplification reactions were conducted using variable amounts of theHIV-1 in vitro transcript, and about 0.07-0.12 pmoles/μl of each primerin 30 μl reaction volumes.

A molecular beacon capable of hybridizing to the HIV-1 amplicons wassynthesized by standard solid-phase phosphite triester chemistry using3′ quencher-linked controlled pore glass (CPG) and 5′fluorophore-labeled phosphoramidite on a Perkin-Elmer (Foster City,Calif.) EXPEDITE model 8909 automated synthesizer. Fluorescein was usedas the fluorophore, DABCYL was used as the quencher, and 2′-methoxynucleotide analogs were used for construction of the molecular beacon.The CPG and phosphoramidite reagents were purchased from Glen ResearchCorporation (Sterling, Va.). Following synthesis, deprotection andcleavage from the solid support matrix, the probes were purified usingpolyacrylamide gel electrophoresis followed by HPLC using standardprocedures that will be familiar to those having an ordinary level ofskill in the art. The target-complementary sequence contained in themolecular beacon, allowing for the substitution of a single inosinenucleotide analog at position four, and the substitution of uricil forthymine bases, was TGGGGGGTACAGTGC (SEQ ID NO:31). Notably, thetarget-hybridizing sequences of the molecular beacon and molecular torchhybridization probes described herein were not fully complementary tothe HIV-1 O group nucleic acids, or HIV-1 O group amplicons synthesizedusing the primers disclosed herein. Additionally, the target-hybridizingsequences of these probes were not fully complementary to the nucleicacids or amplicons for HIV-1 M group members represented by subtypes A,E and F. Conversely, these target-hybridizing sequences were fullycomplementary to HIV-1 subtype B amplicons. The overall sequence of themolecular beacon probe used in the procedure was given by SEQ ID NO:22.

Individual wells in a multiwell plate each contained 30 μl of aTris-buffered solution that included potassium and magnesium salts,N-Acetyl-L-Cysteine, ribonucleotide triphosphates, nucleotidetriphosphates and other reagents, a target polynucleotide, and amolecular beacon. The target polynucleotide was included in amountsranging from 5 to 5×10⁶ copies/reaction. The first-strandpromoter-primer for amplifying the HIV-1 template had thetarget-hybridizing sequence of SEQ ID NO:13 (which was contained withinthe sequence of SEQ ID NO:17). Second-strand primers had the sequence ofeither SEQ ID NO:1 or SEQ ID NO:2. Samples were incubated for 10 minutesat 60° C. to facilitate primer annealing, and then incubated at 42° C.for at least 5 minutes. Aliquots of an enzyme reagent that included bothMMLV reverse transcriptase and T7 RNA polymerase enzymes were added toeach of the tubes using a repeat pipettor. Amplification reactions werecarried out at 42° C., and fluorescence readings were taken every 19.4seconds using a CHROMO4 REAL-TIME DETECTOR (MJ Research; Reno, Nev.), orevery 30 seconds using an OPTICON 2 (MJ Research; Reno, Nev.) real-timeinstrument essentially according to the manufacturer's instructions.Reactions were performed in replicates of 4-8.

The results presented in FIG. 2 showed how the substitution of oneamplification primer for another dramatically increased the quantitativecapacity and precision of the assay. Time-dependent amplificationsignals obtained using a first-strand promoter-primer that included thetarget-hybridizing sequence of SEQ ID NO:13 and a second-strand primerhaving the target-complementary sequence SEQ ID NO:1 showed reducedprecision, as judged by the increased spread among individual datapoints, and substantial divergence from linearity when the level oftemplate used in the reaction fell below 500 copies. This primercombination was preferred for assays intended to quantify HIV-1 nucleicacids at levels greater than 500 copies/reaction. Conversely,time-dependent amplification signals obtained using a first-strandprimer that included the target-complementary sequence SEQ ID NO:13 anda second-strand primer having the target-complementary sequence of SEQID NO:2 showed improved precision and excellent linearity over the rangeof from 25 to 5×10⁶ copies/reaction of the nucleic acid template. Thislatter primer combination advantageously enhanced the low-endquantitative capacity of the assay by 20 fold, a dramatic result thatcould not have been predicted in advance of this showing.

Although not illustrated in FIG. 2, there was a failure to amplify whenusing a first-strand promoter-primer having the target-complementarysequence SEQ ID NO:13 and a second-strand primer having thetarget-complementary sequence SEQ ID NO:1 when using the HIV-1 O grouptemplate at levels less than or equal to 50,000 copies/reaction. Thisdemonstrated that the observed loss of precision at low levels of inputtemplate was a characteristic of the primer combination, and wasindependent of the template being amplified. Conversely, the resultspresented in the following Example confirmed that the combination of afirst-strand promoter-primer having the target-hybridizing sequence ofSEQ ID NO:13 and a second-strand primer having the sequence of SEQ IDNO:2 advantageously gave linear relationships between input templateamounts and the time-dependent amplification signals over an extendedrange with good precision for templates representing multiple HIV-1subtypes.

Example 2 demonstrates that HIV-1 M group, subtype B and HIV-1 O grouptemplates could amplify with good precision over an input template rangethat extended from 25 to 5×10⁵ copies/reaction. Notably, the twotemplates amplified with somewhat different kinetics.

Example 2 Different Kinetic Profiles Characterize Amplification of HIV-1Variants

Amplification reactions were performed essentially as described underExample 1 with the following modifications. Parallel reactions wereconducted using a first-strand promoter-primer having thetarget-hybridizing sequence of SEQ ID NO:13, a second-strand primerhaving the target-complementary sequence of SEQ ID NO:2, and variableamounts of either the subtype B template described in Example 1, or an Ogroup template that included the sequence

(SEQ ID NO: 32)AGTGGGTTCATAGAAGCAGAAGTGATACCAGCAGAAACAGGACAAGAAACTGCCTACTTCCTGTTAAAACTGGCTGCAAGATGGCCTGTTAAAGTAATACATACAGACAACGGGCCTAATTTTACAAGTACAACTATGAAGGCTGCATGTTGGTGGGCCAACATACAACATGAGTTTGGAATACCATATAATCCACAAAGTCAAGGAGTAGTAGAAGCCATGAATAAGGAATTAAAATCAATTATACAGCAGGTGAGGGACCAAGCAGAACACTTAAGAACAGCAGTACAAATGGCAGTATTTGTTCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACACTGCAGGAGAAAGGATAATAGACATATTAGCATCACAAATACAAACAACAGAATTACAAAAACAAATTTTAAAANTTCACAAATTTCGGGTCTATTACAGAGACAGCAGAGACCCTAT.Like the subtype B template, the O group template was also an in vitrotranscript prepared using materials and procedures that will be familiarto those having an ordinary level of skill in the art. Templates wereincluded in the reactions in amounts ranging from 50 to 5×10⁵copies/reaction.

The results presented in FIG. 3 confirmed that advantages associatedwith the combination of a first-strand primer that included thetarget-hybridizing sequence of SEQ ID NO:13 and a second-strand primerhaving the target-complementary sequence of SEQ ID NO:2 extended toamplification of HIV-1 O group templates. More specifically, reactionsconducted using this primer combination advantageously exhibited goodprecision among data points at low levels of HIV-1 template, andlinearity of the time-dependent amplification signal. Moreover, thebeneficial characteristics of this primer combination were observed forboth HIV-1 subtype B and O group templates. As indicated above, testsconducted using the HIV-1 O group template, a first-strandpromoter-primer having the target-hybridizing sequence SEQ ID NO:13 anda second-strand primer having the target-complementary sequence SEQ IDNO:1 failed to yield useful amplification signals when the number ofinput copies of template was below 50,000 copies.

Interestingly, the different HIV-1 template species used in theprocedure gave rise to substantially parallel lines on the graph shownin FIG. 3, with the HIV-1 O group template yielding somewhat sloweramplification kinetics. For example, a reaction conducted using 5,000copies of the HIV-1 subtype B template required about 15 minutes toachieve a positive result, but a similar reaction conducted using theHIV-1 O group template required an additional four minutes to achievethe same result. In a quantitative assay that measures the time toachieve a positive result, such a difference conceivably couldcompromise interpretation of the results and lead to an erroneousconclusion.

Despite the benefits of the primer combination used in this Example, theresults indicated that different HIV-1 variants amplified with differentkinetic profiles. In the instance illustrated in FIG. 3, detection of apositive amplification signal at 15 minutes would ambiguously indicatethe presence of 5,000 copies of the subtype B template, or 500,000copies of the O group template. A desire to perform assays using asingle calibrator, or set of calibrators for quantifying multiple HIV-1species in a single reaction rendered preferable a close relationshipbetween the amplification profiles of the HIV-1 variants to be detected.Thus, to improve the quantitative capacity of the assay even further,reaction conditions were sought to normalize amplification kinetics fordifferent HIV-1 subtypes.

The following Example discloses amplification primers containingmismatches to both HIV-1 subtype B and HIV-1 O group templates, and useof these primers to normalize the amplification kinetics of HIV-1variants. The approach used in this procedure was to substitutenucleotides within the sequence of the first-strand primer such that thesubstitution was complementary to a position contained in the O grouptemplate, yet non-complementary to the sequence contained in the subtypeB template. The object of this approach was to enhance the amplificationkinetics of the O group template relative to the subtype B template.

Example 3 describes methods that identified a first-strand primer whichenhanced amplification kinetics of HIV-1 O group templates. Contrary towhat might have been expected, there was substantially no effect on theamplification kinetics for the HIV-1 subtype B template.

Example 3 Enhancement of Amplification Kinetics of HIV-1 O GroupTemplates

Parallel sets of amplification reactions were prepared to compare theeffects of two different primer combinations on the kinetics ofamplification of HIV-1 subtype B and HIV-1 O group templates. In eachinstance, a first-strand promoter-primer having the target-hybridizingsequence of SEQ ID NO:13 or SEQ ID NO:15 was used in combination with asecond-strand primer having the target-complementary sequence of SEQ IDNO:2. Notably, the sequence of SEQ ID NO:15 differed from the sequenceof SEQ ID NO:13 by the substitution of adenine for thymidine at position15 in the target-hybridizing portion of the primer. This substitutioncorresponds to position 41 of the promoter-primers identified by SEQ IDNO:19 and SEQ ID NO:17. Amounts of HIV-1 templates used in the reactionsranged from 5 to 5×10⁴ copies/reaction. Amplification reactions wereprepared and monitored using materials and procedures essentially asdescribed above.

The results presented in FIGS. 4A-4B indicated that substitution of theprimer having the target-hybridizing sequence of SEQ ID NO:15 for theprimer having the target-hybridizing sequence of SEQ ID NO:13 haddifferent effects on the amplification kinetics of the differenttemplates. More specifically, FIG. 4A shows that the different primersets amplified the HIV-1 subtype B template with substantially identicalkinetics. However, FIG. 4B shows that the HIV-1 O group templateamplified with somewhat more rapid kinetics over the full range of inputtemplate values tested when using the primer having thetarget-hybridizing sequence of SEQ ID NO:15 instead of SEQ ID NO:13.Accordingly, the combination of primers that included thetarget-hybridizing sequences SEQ ID NO:15 and SEQ ID NO:2 advantageouslyamplified the different HIV-1 template species with kinetics that moreclosely approximated each other when compared with the combination ofprimers that included the target-hybridizing sequences SEQ ID NO:13 andSEQ ID NO:2.

In a related procedure, the different primer combinations were used toamplify independent templates representing HIV-1 subtypes A-C, E-F, G/A,H and the HIV-1 O group. The time required to yield a positiveamplification signal was determined for input levels of templateequaling 1,000 copies/reaction. Reactions were performed usingreplicates of six.

The bar graphs in FIGS. 5A-5B demonstrated that the new primercombination advantageously reduced differences between the times neededto achieve positive amplification results for numerous HIV-1 subtypes.FIG. 5A indicates that 3.4 minutes distinguished the times to achievepositive amplification results for the subtype B and O group templateswhen using primers that included the target-hybridizing sequences of SEQID NO:13 and SEQ ID NO:2. In contrast, FIG. 5B shows that thisdifference was reduced to only 1.3 minutes when the primers included thetarget-hybridizing sequences of SEQ ID NO:15 and SEQ ID NO:2.Additionally, differences between the times needed to achieve positiveresults for several subtypes also appeared to be minimized in reactionsconducted using these primers. Despite these improvements, theamplification kinetics for the HIV-1 subtype E and subtype F templatesappeared somewhat retarded compared with the amplification kineticsobserved for the other samples.

It also was shown that, when paired with the primer of SEQ ID NO:2, theprimer having the target-hybridizing sequence of SEQ ID NO:16advantageously improved accuracy of quantitation for the HIV-1 O grouptemplate when compared with the combination of the primers having thetarget-hybridizing sequences of SEQ ID NO:2 and SEQ ID NO:13. Thus, theprimer represents a preferred embodiment of the invention, particularlywhen paired with the primer of SEQ ID NO:2.

Finally, each of the primers identified by SEQ ID NOs:3-6 and 8-12 inTable 1, when paired with the primer of SEQ ID NO:15, and when comparedwith results obtained using the primer of SEQ ID NO:2 in combinationwith the primer of SEQ ID NO:15, behaved substantially equivalently.This pattern was demonstrated using the molecular beacon disclosedherein with the primers from Table 1 identified by SEQ ID NOs:3-9, andusing the molecular torch disclosed herein with the primers from Table 1identified by SEQ ID NO:5 (see below), SEQ ID NO:8, and SEQ IDNOs:10-12. For a reason that is unclear, equally good results were notachieved using the combination of primers having the target-hybridizingsequences of SEQ ID NO:7 and SEQ ID NO:15. Thus, any combination of theprimer of SEQ ID NO:15 with any of the primers identified by SEQ IDNOs:3-6 and 8-12 represents a preferred combination of primers foramplifying HIV-1 nucleic acids. These combinations are particularlypreferred when further combined with a molecular beacon hybridizationprobe, or with a molecular torch hybridization probe.

The foregoing procedures identified a primer combination thatadvantageously was capable of amplifying several HIV-1 subtypes withsubstantially equivalent kinetic profiles. Notably, HIV-1 subtypes E andF exhibited somewhat delayed amplification kinetics compared with theother targets used in the testing procedure. Having already modified thefirst- and second-strand primers, a different approach investigated theeffects of modifying the detection probe used in the fluorescentmonitoring protocol.

Example 4 describes procedures that identified oligonucleotide primersand a probe that yielded substantially equivalent amplification kineticsfor all of the different HIV-1 variants.

Example 4 Time-Dependent Monitoring of Amplicon Synthesis Using aMolecular Torch

Parallel amplification reactions were prepared essentially as describedin the preceding Examples with the following modifications. Afirst-strand primer having the target-hybridizing sequence of SEQ IDNO:15 positioned downstream from a T7 promoter sequence (i.e., thepromoter-primer of SEQ ID NO:19) was used in combination with asecond-strand primer having the sequence SEQ ID NO:5. Additionally, amolecular torch having the sequence of SEQ ID NO:24 (i.e., having thetarget-hybridizing sequence given by SEQ ID NO:23) was substituted forthe molecular beacon having the sequence of SEQ ID NO:22. The moleculartorch was labeled at its 5′ end with a fluorescein fluorophore, and atits 3′ end with a DABCYL quencher moiety. Finally, in vitro transcriptsrepresenting HIV-1 subtypes A-C, E-F, G/A, H and O group were used astemplates at 50 and 1,000 copies/reaction.

A standard curve was prepared from data obtained in trials conductedusing the HIV-1 subtype B templates as illustrative HIV-1 M groupstandards at 50 and 1,000 copies/reaction. Reactions were carried out inreplicates of six. The time required to effect detectable levels ofamplification above background were plotted on the y-axis, and thenumber of copies/reaction of the standard plotted on the x-axis of thestandard curve. The average time required to effect detectable levels ofamplification in each reaction performed using the different HIV-1subtypes was determined, and those time values used to establish averagelog₁₀ copy values by comparison with the standard curve.

The results presented in FIGS. 6A-6B showed that all of the HIV-1variants advantageously were amplified with substantially equalefficiency when using the specified combination of amplificationprimers, and when a molecular torch was substituted for the molecularbeacon hybridization probe. The maximum difference among the timesrequired to achieve positive amplification signals at the 1,000copy/reaction level was reduced to only 1.3 minutes (0.7 log₁₀copies/reaction). Indeed, the difference between the determined numberof HIV-1 subtype F templates (i.e., the species exhibiting the slowestamplification kinetics among the HIV-1 M group) did not exceed 0.7 log₁₀copies/reaction when reactions were initiated using 1,000 templatecopies/reaction. Likewise, the determined number of HIV-1 O grouptemplates differed from the actual number of template copies/reaction byno more than 0.5 log₁₀ copies/reaction when reactions were initiatedusing 1,000 template copies/reaction. The nature of real-timeamplification systems, such as those disclosed herein, gives improvedprecision at increasing copy levels. Accordingly, differences betweenthe actual number of HIV-1 template copies/reaction and the determinednumber of template copies/reaction will be less than 0.7 log₁₀copies/reaction for reactions carried out using greater than 1,000copies/reaction of the HIV-1 subtype F species, and will be less than0.5 log₁₀ copies/reaction for reactions carried out using greater than1,000 copies/reaction of the HIV-1 O group template.

The fact that the different HIV-1 subtypes gave more normalized timevalues in this procedure was attributed to the substitution of amolecular torch for a molecular beacon (as illustrated in the previousExample), because it was independently established that thesecond-strand primers of SEQ ID NO:2 and SEQ ID NO:5 performedessentially equivalently in the amplification reactions. Notably, thetarget-hybridizing sequences of SEQ ID NO:2 and SEQ ID NO:5 both conformto the consensus CCACAATTTTRAAAGAAAAGGG (SEQ ID NO:33). Further, ourfinding demonstrates that molecular torches can have advantages overmolecular beacons when used as probes for real-time monitoring ofisothermal amplification reactions, particularly when the target bindingportion of the probe is required to hybridize to amplicons that are notfully complementary. As indicated above, the target-hybridizing sequenceof the molecular torch was not fully complementary to the HIV-1 O groupnucleic acid or amplicon, or to the nucleic acids or amplicons of HIV-1M group subtypes A, E and F. Although not shown in the figure, all ofthe different subtypes were easily detected when present at the level of50 copies/reaction, thereby demonstrating robustness of theamplification system.

Using a combination of primers and a probe that amplify HIV-1 M groupand HIV-1 O group nucleic acids with substantially equal efficiency in areal-time amplification protocol, it is preferred to employ apolynucleotide of a single HIV-1 subtype as a calibration standard forassays capable of quantifying numerous different HIV-1 subtypes. Forexample, it is preferred to use an HIV-1 M group standard, such as aknown amount of an HIV-1 subtype B nucleic acid, as a calibrationstandard. This HIV-1 M group nucleic acid standard can be used forestablishing a point on a standard curve, and the resulting standardcurve can be used for quantifying both HIV-1 M group and HIV-1 O groupnucleic acids. Of course, it is also possible to employ a collection ofHIV-1 M group standards, each having a different known amount of HIV-1 Mgroup nucleic acids, to establish several points on a standard curve,and to use the resulting standard curve for quantifying the variousHIV-1 M group and O group nucleic acids. Alternatively, instead of usingthe HIV-1 M group nucleic acid standard, HIV-1 O group standards can beemployed instead. In this instance known amounts of an HIV-1 O groupnucleic acid are employed as standards to create a standard curve byamplifying the nucleic acids using a combination of amplificationprimers and hybridization probe that amplify the HIV-1 M group and HIV-1O group nucleic acids with substantially equal efficiencies. Theresulting standard curve can be used for quantifying both HIV-1 O groupand M group nucleic acids. It is even contemplated that a chimericstandard nucleic acid which is not strictly an HIV-1 M group nucleicacid or an HIV-1 O group nucleic acid could be used as a standard forquantifying both HIV-1 M group and O group nucleic acids.

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

1. A method of quantifying HIV-1 M group nucleic acids and HIV-1 O groupnucleic acids in a test sample that comprises nucleic acids, said methodcomprising the steps of: (a) contacting nucleic acids of the test samplewith a first amplification primer, a second amplification primer, and amolecular torch hybridization probe, the base sequence of the moleculartorch hybridization probe being CGGIGGGUACAGUGCCCCCG (SEQ ID NO:24); (b)amplifying, in an isothermal in vitro nucleic acid amplificationreaction, any HIV-1 M group and HIV-1 O group nucleic acids that may bepresent in the test sample using said first and second amplificationprimers; (c) monitoring, with the molecular torch hybridization probe,the time-dependent production of amplification products in theisothermal in vitro nucleic acid amplification reaction to determine atime-dependent value indicative of the combined starting quantity ofHIV-1 M group and HIV-1 O group nucleic acids present in the isothermalin vitro nucleic acid amplification reaction; and (d) quantifying thecombined amounts of HIV-1 M group and HIV-1 O group nucleic acidspresent in the test sample using a standard curve and the time-dependentvalue determined in step (c).
 2. The method of claim 1, wherein thestandard curve used in step (d) relates pre-amplification amounts of anHIV-1 standard polynucleotide, and the time required to achieve athreshold level of amplification for the HIV-1 standard polynucleotidein the isothermal in vitro nucleic acid amplification reaction, andwherein the difference between the number of starting copies perreaction quantified in step (d) and the actual number of starting copiesper reaction is no greater than 0.5 log₁₀ copies per reaction forindependent isothermal in vitro amplification reactions conducted usingHIV-1 M group subtype-B nucleic acid templates, and HIV-1 O grouptemplates, each at actual starting levels of 1,000 copies per reaction.3. The method of claim 2, wherein the HIV-1 standard polynucleotide isan HIV-1 subtype B nucleic acid.
 4. The method of claim 1, wherein thefirst amplification primer in step (a) is a promoter-primer thatconsists of a phage promoter sequence joined upstream of SEQ ID NO:15,wherein the isothermal in vitro nucleic acid amplification reaction instep (b) is a transcription mediated amplification reaction thatsynthesizes RNA amplification products, and wherein step (c) comprisesmonitoring, with the molecular torch hybridization probe, thetime-dependent production of RNA amplification products in thetranscription mediated amplification reaction.
 5. The method of claim 4,wherein the standard curve used in step (d) relates pre-amplificationamounts of an HIV-1 standard polynucleotide, and the time required toachieve a threshold level of amplification for the HIV-1 standardpolynucleotide in the isothermal in vitro nucleic acid amplificationreaction, and wherein the HIV-1 standard polynucleotide comprises eitheran HIV-1 M group standard or an HIV-1 O group standard.
 6. The method ofclaim 4, wherein the second amplification primer in step (a) is selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
 7. The methodof claim 4, wherein the second amplification primer in step (a) is SEQID NO:2.
 8. The method of claim 4, wherein the second amplificationprimer in step (a) is SEQ ID NO:5.
 9. The method of claim 8, wherein thestandard curve used in step (d) relates pre-amplification amounts of anHIV-1 standard polynucleotide, and the time required to achieve athreshold level of amplification for the HIV-1 standard polynucleotidein the isothermal in vitro nucleic acid amplification reaction, andwherein the HIV-1 standard polynucleotide is an HIV-1 M group standard,and not an HIV-1 O group standard.
 10. The method of claim 9, whereinthe difference between the number of starting copies per reactionquantified in step (d) and the actual number of starting copies perreaction is no greater than 0.5 log₁₀ copies per reaction forindependent isothermal in vitro amplification reactions conducted usingHIV-1 M group subtype-B nucleic acid templates, and HIV-1 O grouptemplates, each at actual starting levels of 1,000 copies per reaction.11. The method of claim 9, wherein the HIV-1 M group standard is anHIV-1 subtype B nucleic acid.
 12. The method of claim 1, wherein thestandard curve used in step (d) relates pre-amplification amounts of anHIV-1 standard polynucleotide, and the time required to achieve athreshold level of amplification for the HIV-1 standard polynucleotidein the isothermal in vitro nucleic acid amplification reaction, andwherein the HIV-1 standard polynucleotide in step (d) comprises eitheran HIV-1 M group standard or an HIV-1 O group standard.
 13. The methodof claim 12, wherein the HIV-1 M group standard is an HIV-1 subtype Bnucleic acid.
 14. The method of claim 13, wherein the difference betweenthe number of starting copies per reaction quantified in step (d) andthe actual number of starting copies per reaction is no greater than 0.5log₁₀ copies per reaction for independent isothermal in vitroamplification reactions conducted using HIV-1 M group subtype-B nucleicacid templates, and HIV-1 O group templates, each at actual startinglevels of 1,000 copies per reaction.
 15. The method of claim 14, whereinthe first amplification primer in step (a) is a promoter-primer thatconsists of a phage promoter sequence joined upstream of SEQ ID NO:15,wherein the isothermal in vitro nucleic acid amplification reaction instep (b) is a transcription mediated amplification reaction thatsynthesizes RNA amplification products, and wherein step (c) comprisesmonitoring, with the molecular torch hybridization probe, thetime-dependent production of RNA amplification products in thetranscription mediated amplification reaction.
 16. The method of claim1, wherein the molecular torch hybridization probe of step (a) comprisesa fluorophore moiety and a quencher moiety, and wherein step (c)comprises monitoring a fluorescent signal generated by the fluorophoremoiety.
 17. The method of claim 16, wherein nucleotide positions 15 and16 of the molecular torch hybridization probe are joined by anon-nucleotide linker, and wherein a fluorescein moiety is joined at oneterminus of SEQ ID NO:24.
 18. The method of claim 1, wherein saidstandard curve is stored electronically in a memory device of a testinginstrument.
 19. The method of claim 1, wherein the difference betweenthe number of starting copies per reaction quantified in step (d) andthe actual number of starting copies per reaction is no greater than 0.5log₁₀ copies per reaction for independent isothermal in vitroamplification reactions conducted using HIV-1 M group subtype-B nucleicacid templates, and HIV-1 O group templates, each at actual startinglevels of 1,000 copies per reaction.
 20. The method of claim 1, whereinnucleotide positions 15 and 16 of the molecular torch hybridizationprobe are joined by a non-nucleotide linker, and wherein a fluoresceinmoiety is joined at one terminus of SEQ ID NO:24.